Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

Ian G. Jennings, Richard G.H. Cotton, Bostjan Kobe

Research output: Contribution to journalArticleResearchpeer-review

8 Citations (Scopus)

Abstract

The 3-dimensional structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the Km for substrate or cofactor. We conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. In contrast the PAH mutant, S349T, showed an 18-fold increase in Km for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate Km, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different.

Original languageEnglish
Pages (from-to)238-244
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume384
Issue number2
DOIs
Publication statusPublished - 15 Dec 2000
Externally publishedYes

Keywords

  • Catalytic site
  • In vitro mutagenesis
  • Phenylalanine binding site
  • Phenylalanine hydroxylase
  • Tetrahydrobiopterin

Cite this

@article{b8b96fbc38fb4a3aa98d37f1631bd50d,
title = "Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site",
abstract = "The 3-dimensional structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the Km for substrate or cofactor. We conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. In contrast the PAH mutant, S349T, showed an 18-fold increase in Km for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate Km, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different.",
keywords = "Catalytic site, In vitro mutagenesis, Phenylalanine binding site, Phenylalanine hydroxylase, Tetrahydrobiopterin",
author = "Jennings, {Ian G.} and Cotton, {Richard G.H.} and Bostjan Kobe",
year = "2000",
month = "12",
day = "15",
doi = "10.1006/abbi.2000.2111",
language = "English",
volume = "384",
pages = "238--244",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Elsevier",
number = "2",

}

Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site. / Jennings, Ian G.; Cotton, Richard G.H.; Kobe, Bostjan.

In: Archives of Biochemistry and Biophysics, Vol. 384, No. 2, 15.12.2000, p. 238-244.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

AU - Jennings, Ian G.

AU - Cotton, Richard G.H.

AU - Kobe, Bostjan

PY - 2000/12/15

Y1 - 2000/12/15

N2 - The 3-dimensional structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the Km for substrate or cofactor. We conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. In contrast the PAH mutant, S349T, showed an 18-fold increase in Km for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate Km, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different.

AB - The 3-dimensional structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the Km for substrate or cofactor. We conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. In contrast the PAH mutant, S349T, showed an 18-fold increase in Km for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate Km, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different.

KW - Catalytic site

KW - In vitro mutagenesis

KW - Phenylalanine binding site

KW - Phenylalanine hydroxylase

KW - Tetrahydrobiopterin

UR - http://www.scopus.com/inward/record.url?scp=0034671776&partnerID=8YFLogxK

U2 - 10.1006/abbi.2000.2111

DO - 10.1006/abbi.2000.2111

M3 - Article

VL - 384

SP - 238

EP - 244

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -